Summary The ultimate goal of this research is to provide integrated systems that allow complex biological samples to be analyzed with high sensitivity and broad dynamic range. These systems are applicable to proteins, DNA and RNA oligomers, and metabolites from bodily fluids and tissues. The proposed project is specifically focused on developing novel sample plates employing collimated hole structures that allow a variety of separation techniques to be efficiently coupled to MALDI-TOF and TOF-TOF mass spectrometry. The first specific aim for the proposed research is an improved interface between HPLC and MALDI MS-MS that is compatible with 1 mm ID columns, operating at flow rates up to 50 ?L/min without sacrificing sensitivity for trace components. This will allow larger sample loadings and is expected to increase the dynamic range by at least a factor of fifty and allow faster, high-resolution separations. The second specific aim is to develop a direct interface between slab gel electrophoresis and MALDI-TOF-TOF that allows extraction, digestion, and concentration of samples on the MALDI plate with minimal sample loss and no loss in spatial resolution. A third specific aim is to develop a similar interface directly with frozen tissue samples both for imaging the intact proteins present, and for digesting and identifying the proteins by MALDI-TOF MS-MS. This project addresses a major bottleneck limiting the utility of mass spectrometry as an essential tool needed for truly global analyses of biological systems. It is now generally recognized that mass spectrometry interfaced with efficient separation technologies is essential for large-scale analyses of complex biological samples in functional proteomics and metabolomics; but currently available systems are inadequate. Improved MALDI-TOF and TOF-TOF mass spectrometry can provide the MS performance needed, but improved interfaces with separations are equally important. This Phase I project will establish the feasibility of a new approach to MALDI that allows parallel processing of large numbers of samples in a novel micro- fluidic system to achieve high throughput and broad dynamic range. A future Phase II effort will focus on developing practical systems that can be used by biologists in a variety of research applications. These will be developed and evaluated working with collaborators directly involved in such research. [unreadable] Recent advances in MALDI-TOF and TOF-TOF technology make this approach an attractive alternative to the established electrospray LC-MS-MS systems for quantitation and identification of proteins and peptides in complex biological samples. Despite some early success, adoption has been slow, probably because the potential advantages have not yet been clearly demonstrated. This project, together with improved MALDI-TOF-TOF instrumentation being developed in a separate project, will demonstrate the real advantages of this new approach. [unreadable] [unreadable] [unreadable]